Spectroscopic sensing and quantification of AP-endonucleases using fluorescence-enhancement by cis- trans isomerization of cyanine dyes

JunHo Cho; Sanghoon Oh; DongHun Lee; Jae Won Han; Jungmin Yoo; Daeho Park; Gwangrog Lee

doi:10.1039/d0ra08051a

Spectroscopic sensing and quantification of AP-endonucleases using fluorescence-enhancement by cis- trans isomerization of cyanine dyes

RSC Adv. 2021 Mar 18;11(19):11380-11386. doi: 10.1039/d0ra08051a. eCollection 2021 Mar 16.

Authors

JunHo Cho¹, Sanghoon Oh², DongHun Lee¹, Jae Won Han¹, Jungmin Yoo¹, Daeho Park^{1

3}, Gwangrog Lee^{1

2

3}

Affiliations

¹ School of Life Sciences, Gwangju Institute of Science and Technology Gwangju 500-712 Korea glee@gist.ac.kr +82-62-715-2484.
² Department of Biomedical Science and Engineering, Gwangju Institute of Science and Technology Gwangju 500-712 Korea.
³ Cell Mechanobiology Research Center, Gwangju Institute of Science and Technology Gwangju 61005 Republic of Korea.

Abstract

Apurinic/apyrimidinic (AP) endonucleases are vital DNA repair enzymes, and proposed to be a prognostic biomarker for various types of cancer in humans. Numerous DNA sensors have been developed to evaluate the extent of nuclease activity but their DNA termini are not protected against other nucleases, hampering accurate quantification. Here we developed a new fluorescence enhancement (FE)-based method as an enzyme-specific DNA biosensor with nuclease-protection by three functional units (an AP-site, Cy3 and termini that are protected from exonucleolytic cleavage). A robust FE signal arises from the fluorescent cis-trans isomerization of a cyanine dye (e.g., Cy3) upon the enzyme-triggered structural change from double-stranded (ds)DNA to single-stranded (ss)DNA that carries Cy3. The FE-based assay reveals a linear dependency on sub-nanomolar concentrations as low as 10^-11 M for the target enzyme and can be also utilized as a sensitive readout of other nuclease activities.